Alpha-hemolysin variants

ABSTRACT

Described herein are engineered alpha-hemolysin subunits having mutated oligomerization domains for assembling into heptameric nanopores in lipid bilayers.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.16/238,874, filed Jan. 3, 2019, which is a continuation of U.S. patentapplication Ser. No. 15/274,770, filed Sep. 23, 2016, which claims thebenefit of U.S. Provisional Application No. 62/232,175, filed Sep. 24,2015 and of U.S. Provisional Application No. 62/244,852, filed Oct. 22,2015, each of which is incorporated herein by reference in theirentireties.

SEQUENCE LISTING

A sequence listing comprising SEQ ID NOS: 1-11 is attached hereto. Eachsequence provided in the sequence listing is incorporated herein byreference, in its entirety, for all purposes. Said ASCII copy, createdon Jun. 30, 2020, is named 04338_544US3_Seq_Listing.txt and is 21kilobytes in size.

TECHNICAL FIELD

Disclosed are compositions and methods relating to Staphylococcal aureusalpha-hemolysin variants, and αHL mutated variants. The alpha-hemolysin(α-HL) variants are useful, for example, as a nanopore in a device fordetermining polymer sequence information. The αHL mutated variants areuseful for manipulating the stoichiometry of subunits to providefunctional heptameric αHL pores. The nanopores, methods and systemsdescribed herein provide quantitative detection of single strand nucleicacids, such as DNA, RNA, etc., employing nanopore-based single-moleculetechnology with improved characteristics.

BACKGROUND

Hemolysins are members of a family of protein toxins that are producedby a wide variety of organisms. Some hemolysins, for example alphahemolysins, can disrupt the integrity of a cell membrane (e.g., a hostcell membrane) by forming a pore or channel in the membrane. Pores orchannels that are formed in a membrane by pore forming proteins can beused to transport certain polymers (e.g., polypeptides orpolynucleotides) from one side of a membrane to the other.

Alpha-hemolysin (α-HL, α-HL, αHL, aHL or alpha-HL) is a self-assemblingtoxin which forms an aqueous channel in the membrane of a host cell.Alpha-HL has become a principal component for the nanopore sequencingcommunity. It has many advantageous properties including high stability,self assembly and a pore diameter which is wide enough to accommodatesingle stranded DNA but not double stranded DNA (Kasianowicz et al.,1996).

Previous work on DNA detection in the a-HL pore has focused on analyzingthe ionic current signature as DNA translocates through the pore(Kasianowicz et al., 1996, Akeson et al., 1999, Meller et al., 2001), avery difficult task given the translocation rate (˜1 nt/μs at 100 mV)and the inherent noise in the ionic current signal. Higher specificityhas been achieved in nanopore-based sensors by incorporation of probemolecules permanently tethered to the interior of the pore (Howorka etal., 2001a and Howorka et al., 2001b; Movileanu et al., 2000).

The wild-type a-HL results in significant number of deletion errors,i.e., bases are not measured. Therefore, α-HL nanopores with improvedproperties are desired.

BRIEF SUMMARY OF THE INVENTION

The invention features a mutant staphylococcal alpha hemolysin (αHL)polypeptide containing an amino acid variation that enhances the time tothread (TTT), e.g., decreases the time to capture of the molecule ofinterest, e.g., relative to the TTT of the parent or wild type αHL.

The presently disclosed variants reduce the time thread of the moleculeof interest, e.g., various tagged nucleotides or a nucleotide to besequenced.

Disclosed herein are α-hemolysin (αHL) variants. The α-hemolysin (αHL)variants are derived from a parental α-HL polypeptide, e.g., SEQ IDNO:3, and comprise one or more mutation(s) relative to the parental α-HLpolypeptide. In some embodiments, the variant includes a substitution ata position corresponding to position 12 or 17 of SEQ ID NO:3 (maturea-HL). In some embodiments, the variant further comprises substitutionH144A. In some embodiments, the substitution comprises one or morepositive charges. In some embodiments, the variant comprises asubstitution at a position corresponding to one or more of residues T12and/or N17. In some embodiments, the variant comprises a substitutionselected from T12K, T12R, N17K, N17R and combinations thereof. In someembodiments, the variant has an altered time to thread (TTT) relative tothe parent α-hemolysin. In some embodiments, the TTT is decreased. Insome embodiments, the variant comprises a substitution at a positioncorresponding to a residue selected from the group consisting of T12R orT12K, and/or N17R or N17K in α-hemolysin (αHL) from Staphylococcusaureus (SEQ ID NOs:1 and 3). In some embodiments, the substitution isT12K. In some embodiments, the substitution is T12R. In someembodiments, the substitution is N17K. In some embodiments, thesubstitution is N17R. In some embodiments, the variant a-HL having analtered characteristic as compared to a parental a-hemolysin (e.g.,AAA26598) comprises H144A and at least one additional mutation selectedfrom

-   -   a. T12K/R;    -   b. N17K/R;        or combinations thereof.

In some embodiments, the amino acid substitution allows the addition ofheterologous molecules, e.g., PEG. In some embodiments, the a-HL varianthas post-translational modifications.

In some embodiments, the substitution is a non-native amino acid that isbasic or positively charged at a pH from about 5 to about 8.5.

In an aspect, there is provided a heptameric pore assembly (e.g.,nanopore assembly) comprising at least one a-hemolysin (αHL) variant asdescribed herein. In one embodiment the invention provides a heteromericpore assembly containing a mutant αHL polypeptide (M), e.g., a poreassembly which contains a wild type (WT) staphylococcal αHL polypeptideand a mutant αHL polypeptide in which an amino acid variant (as providedfor herein) of the mutant αHL polypeptide occupies a position in atransmembrane channel of the pore structure. For example, the ratio ofWT and variant αHL polypeptides is expressed by the formulaWT_(7-n)M_(n), where n is 1, 2, 3, 4, 5, 6, or 7; preferably the ratioof αHL polypeptides in the heteroheptamer is WT_(7-n)M_(n); mostpreferably, the ratio is WT₆M₁. Homomeric pores in which each subunit ofthe heptamer is a mutated αHL polypeptide (i.e., where n=7) are alsoencompassed by the invention. Heptameric pores can be assembled fromconcatemer subunits of at least two linked monomers in combination withconcatemer subunits of at least two linked monomers. Alternatively,heptameric pores can be assembled from a combination of concatemersubunits of at least two linked monomers and individual monomers. Thus,the ratio of WT to variant subunits in heptamers of concatemers ormixtures of concatemers and monomers will depend on the size and numbersof concatemers.

In some instances, a polymerase is associated with the nanopore (e.g.,covalently linked to the nanopore) and the polymerase performsnucleotide incorporation events, i.e., retains enzymatic activity.

In an aspect, there is provided a nucleic acid encoding an a-HL variantas described herein.

In an aspect, there is provided a vector comprising a nucleic acidencoding an alpha-hemolysin variant as described herein.

In an aspect, there is provided a host cell transformed with the vectorcomprising a nucleic acid encoding an alpha-hemolysin variant asdescribed herein.

In an aspect, there is provided a method of producing an alpha-hemolysinvariant comprising the steps of: (a) culturing a host cell comprising anucleic acid encoding an alpha-hemolysin variant as described herein ina suitable culture medium under suitable conditions to producealpha-hemolysin variant; and (b) obtaining said produced alpha-hemolysinvariant.

In an aspect, there is provided a method for detecting a targetmolecule, comprising: (a) providing a chip comprising a nanopore asdescribed herein in a membrane that is disposed adjacent or in proximityto a sensing electrode; (b) directing a nucleic acid molecule throughsaid nanopore, wherein said nucleic acid molecule is associated with areporter molecule, wherein said nucleic acid molecule comprises anaddress region and a probe region, wherein said reporter molecule isassociated with said nucleic acid molecule at said probe region, andwherein said reporter molecule is coupled to a target molecule; (c)sequencing said address region while said nucleic acid molecule isdirected through said nanopore to determine a nucleic acid sequence ofsaid address region; and (d) identifying, with the aid of a computerprocessor, said target molecule based upon a nucleic acid sequence ofsaid address region determined in (c).

In one aspect, there is provided a hetero-oligomeric α-hemolysin (αHL)heptamer comprising at least one preceding and at least one followingoligomerization subunits, each oligomerization subunit comprising atleast one αHL monomer and/or at least one concatemer of αHL monomershaving one or more mutations in a first oligomerization domain, and/orone or more mutations in a second oligomerization domain; wherein atleast one of said mutations on said first and/or second domain is abreaking mutation that prevents self-oligomerization of said at leastone preceding and said at least one following oligomerization subunits.

In another aspect, the hetero-oligomeric αHL heptamer described hereincan further comprise at least one cognate and/or rescue mutation on thefirst oligomerization domain in the at least one precedingoligomerization subunit and/or at least one cognate and/or rescuemutation in the second oligomerization domain in the at least onefollowing oligomerization subunit, wherein the at least one cognateand/or rescue mutation determines inter-subunit contact between the atleast one preceding and the at least one following oligomerizationsubunits to specify the sequence of oligomerization subunits in thehetero-oligomeric αHL heptamer. An example of cognate mutations that canbe made in oligomerization domains of preceding and following subunitsthat enable oligomerization of the subunits is the pair of mutationsH35I and Y101H.

In some aspects, the αHL heptamer can be formed by at least onepreceding oligomerization subunit that is a concatemer of αHL monomers,and at least one following subunit is at least one αHL monomer.

In other aspects, the αHL heptamer can be formed by at least onepreceding oligomerization subunit and at least one followingoligomerization subunit that each are concatemers of αHL monomers.

In yet other aspects, the αHL heptamer can be formed by preceding andfollowing oligomerization subunits that are αHL monomers.

In yet another aspect, the αHL heptamer can be formed by at least onepreceding oligomerization subunit that is a concatemer of αHL monomers,and at least one following subunit that is an αHL monomer.

In yet another aspect, the αHL heptamer can be formed by at least onepreceding oligomerization subunit that is a αHL monomer, and at leastone following subunit that is a concatemer of αHL monomers.

In some cases, the monomers and/or the concatemers of monomers of theαHL heptamer comprise one or more polypeptides of SEQ ID NO:3.

In one aspect, mutations in the first oligomerization domain of an αHLheptamer can be made at positions corresponding to amino acids 2-28,35-42, and 43-61 of SEQ ID NO:3. Examples of mutations that can be madein the first oligomerization domain include amino acid substitutionscorresponding to H35D, H35E, H35I, H35L, D24A, V26D, K375, andD24A+V26D+K37S of SEQ ID NO:3. Mutations in the second oligomerizationdomain can be made at positions corresponding to amino acids 95-104,158-164, and 228-236 of SEQ ID NO:3. Examples of mutations that can bemade in the second oligomerization domain include amino acidsubstitutions corresponding to T233R, S99K, Y101D, Y101H, and T233R+S99Kof SEQ ID NO:3.

In all aspects, the αHL heptamer retains the ability to form a pore in alipid bilayer.

In some cases, the αHL heptamer can further comprise a polymerase thatis attached to one or more of the preceding and/or followingoligomerization subunits. In another aspect, in addition to themutations in the first and/or second oligomerization domains, the αHLheptamer can further comprise an αHL polypeptide comprising an aminoacid substitution at a position corresponding to position 12 or 17 ofSEQ ID NO:3, wherein the substitution comprises one or more positivecharges. The substitution at positions 12 or 17 can be selected fromT12K, T12R, N17K, N17R and combinations thereof. αHL heptamerscomprising αHL polypeptides having substitutions at positions 12 or 17may have an altered time to thread (TTT) relative to the parentα-hemolysin. For example, the TTT can be decreased.

In another aspect, a plurality of polynucleotides encoding at least onepreceding and one following oligomerization subunits of thehetero-oligomeric αHL heptamer described herein, are provided.

In another aspect, provided are host cells transformed or transfectedwith an expression vector encoding one of each of the polynucleotidesencoding the oligomerization subunits of the hetero-oligomeric αHLheptamer.

In another aspect, a method is provided for preparing at least onepreceding and at least one following oligomerization subunits of an αHLheptamer that comprises culturing the host cells transfected ortransformed with polynucleotides encoding the oligomerization subunitsof the hetero-oligomeric αHL heptamer. The method can further compriseisolating the at least one preceding and at least one followingoligomerization subunits of the αHL heptamer from the host cell culture.

In another aspect, provided is a heptameric pore assembly comprising ahetero-oligomeric αHL heptamer as described herein.

Other objects, features and advantages of the present invention willbecome apparent from the following detailed description. It should beunderstood, however, that the detailed description and specificexamples, while indicating preferred embodiments of the invention, aregiven by way of illustration only, since various changes andmodifications within the scope and spirit of the invention will becomeapparent to one skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1-5 each comprise two figures, e.g., FIGS. 1A and 1B. The A figurefor each figure is a histogram of the number of capture events which hada “time-to-thread” equal to the time bin shown on the x-axis. The Bfigure for each figure is a portion of the raw data for thecorresponding figure A.

FIGS. 1A and 1B show the results for the wild-type a-hemolysin nanopore.FIG. 1A (top panel) shows “time-to-thread” data. This data is combinedfrom many pores which were capturing the tagged nucleotides indicatingthe pore had both a polymerase and a template DNA molecule. The mean andmedian values, along with the standard deviation for wild type αHL are20.7 ms, 16.1 ms and 1.5 ms respectively, and the total number ofsquarewaves used for the calculations is 41910.

FIG. 1B (bottom panel) shows some raw data with five consecutivesquarewaves shown. The data points between the solid lines represent theopen channel (where no tagged nucleotide is threaded in the pore) andthe data in-between the dashed lines represents when the taggednucleotide has threaded into the pore and is blocking ions movingthrough the channel. The electrode is cycled between positive andnegative 100 mV, and in our system data points are not recorded when anegative voltage is applied. Thus, all the data points are collectedfrom the positively applied potential, and the time where there is anabsence of data points (between 1716.9-1717 sec for example) is when theelectrodes have a negative voltage applied to them. In this example the“time-to-thread” measurement is calculated from squarewaves which have athreaded level observable, and, the previous squarewave had a threadedlevel at the end of the positive voltage (indicating that the tag wasthreaded in the pore and bound by the polymerase).

FIGS. 2A and 2B show the results for the a-hemolysin nanopore comprisinga T12K mutation. FIG. 2A (top panel) is data combined from many poreswhich were capturing the tagged nucleotides indicating the pore had botha polymerase and a template DNA molecule. The mean and median values,along with the standard deviation for T12K αHL are 19.7 ms, 14.5 ms and1.5 ms respectively, and the total number of squarewaves used for thecalculations is 4311.

FIG. 2B (bottom panel) shows some raw data with five consecutivesquarewaves shown. The data points between the solid lines represent theopen channel (where no tagged nucleotide is threaded in the pore) andthe data in-between the dashed lines represents when the taggednucleotide has threaded into the pore and is blocking ions movingthrough the channel. The electrode is cycled between positive andnegative 100 mV, and in our system data points are not recorded when anegative voltage is applied. Thus, all the data points are collectedfrom the positively applied potential, and the time where there is anabsence of data points (between 1600.4-1601.2 sec for example) is whenthe electrodes have a negative voltage applied to them. In this examplethe “time-to-thread” measurement is calculated from squarewaves whichhave a threaded level observable, and, the previous squarewave had athreaded level at the end of the positive voltage (indicating that thetag was threaded in the pore and bound by the polymerase).

FIGS. 3A and 3B show the results for the α-hemolysin nanopore comprisinga T12R mutation. FIG. 3A is data combined from many pores which werecapturing the tagged nucleotides indicating the pore had both apolymerase and a template DNA molecule. The mean and median values,along with the standard deviation for T12R αHL are 16.9 ms, 10.5 ms and1.5 ms respectively, and the total number of squarewaves used for thecalculations is 4138.

FIG. 3B (bottom panel) shows some raw data with five consecutivesquarewaves shown. The data points between the solid lines represent theopen channel (where no tagged nucleotide is threaded in the pore) andthe data in-between the dashed lines represents when the taggednucleotide has threaded into the pore and is blocking ions movingthrough the channel. The electrode is cycled between positive andnegative 100 mV, and in our system data points are not recorded when anegative voltage is applied. Thus, all the data points are collectedfrom the positively applied potential, and the time where there is anabsence of data points (between 267.2-268.2 sec for example) is when theelectrodes have a negative voltage applied to them. In this example the“time-to-thread” measurement is calculated from squarewaves which have athreaded level observable, and, the previous squarewave had a threadedlevel at the end of the positive voltage (indicating that the tag wasthreaded in the pore and bound by the polymerase).

FIGS. 4A and 4B show the results for the a-hemolysin nanopore comprisinga N17R mutation. FIG. 4A (top panel) is data combined from many poreswhich were capturing the tagged nucleotides indicating the pore had botha polymerase and a template DNA molecule. The mean and median values,along with the standard deviation for N17R αHL are 17.5 ms, 10.5 ms and1.7 ms respectively, and the total number of squarewaves used for thecalculations is 3877.

FIG. 4B (bottom panel) shows some raw data with five consecutivesquarewaves shown. The data points between the solid lines represent theopen channel (where no tagged nucleotide is threaded in the pore) andthe data in-between the dashed lines represents when the taggednucleotide has threaded into the pore and is blocking ions movingthrough the channel. The electrode is cycled between positive andnegative 100 mV, and in our system data points are not recorded when anegative voltage is applied. Thus, all the data points are collectedfrom the positively applied potential, and the time where there is anabsence of data points (between 344-344.9 sec for example) is when theelectrodes have a negative voltage applied to them. In this example the“time-to-thread” measurement is calculated from squarewaves which have athreaded level observable, and, the previous squarewave had a threadedlevel at the end of the positive voltage (indicating that the tag wasthreaded in the pore and bound by the polymerase).

FIGS. 5A and 5B show the results for the a-hemolysin nanopore comprisinga N17K mutation. FIG. 5A (top panel) shows combined data from many poreswhich were capturing the tagged nucleotides indicating the pore had botha polymerase and a template DNA molecule. The mean and median values,along with the standard deviation for N17K αHL are 5.7 ms, 2.4 ms and0.7 ms respectively, and the total number of squarewaves used for thecalculations is 2424.

FIG. 5B (bottom panel) shows some raw data with five consecutivesquarewaves shown. The data points above the solid line represent theopen channel (where no tagged nucleotide is threaded in the pore) andthe data in-between the dashed lines represents when the taggednucleotide has threaded into the pore and is blocking ions movingthrough the channel. The electrode is cycled between positive andnegative 100mV, and in our system data points are not recorded when anegative voltage is applied. Thus, all the data points are collectedfrom the positively applied potential, and the time where there is anabsence of data points (between 79.5-80.5 sec for example) is when theelectrodes have a negative voltage applied to them. In this example the“time-to-thread” measurement is calculated from squarewaves which have athreaded level observable, and, the previous squarewave had a threadedlevel at the end of the positive voltage (indicating that the tag wasthreaded in the pore and bound by the polymerase).

FIG. 6A shows a diagram of an αHL subunit (600) having a first (601)oligomerization domain (□) in a first region of the subunit, and asecond (602) oligomerization domain (

) in a second region of the subunit. The oligomerization subunit shownis a monomer subunit.

FIG. 6B shows a diagram of a hetero-oligomeric αHL heptamer of 7different oligomerization subunits, which in this instance are monomersi, ii, iii, iv, v, vi, and vii. Interactions between firstoligomerization domains on preceding subunits (□) and secondoligomerization domains on following subunits (

) are depicted (ζ→).

FIG. 6C shows a diagram of subunits comprising mutations that enableinter-subunit interactions (shown as solid double arrow lines), andmutations that inhibit inter-subunit interactions (shown as crosseddashed double arrow lines). A subunit having breaking mutation 1 (BM1)does not interact with a wild type subunit (WT) or with a subunit havinga breaking mutation 2 (BM2). Subunits having rescue mutations (RM) andcognate mutations (CM), can interact with a subunit having a breakingmutation, e.g., breaking mutation 1. Subunits having rescue mutations(RM) can also interact with subunits that are wild type (WT).

FIG. 6D shows a diagram of an αHL concatemer subunit of two monomers.The first oligomerization domain of the concatemer subunit (□) ispresent on a first subunit (shown here as vii), and the secondoligomerization domain of the concatemer subunit (

) is present on a second subunit (shown here as i).

FIGS. 7A-7F show diagrams of concatemers of two (7A), three (7B, 7C) andfour (7D, 7E, 7F) αHL monomers joined by linkers (

), e.g., (GS)₅ (SEQ ID NO: 9). Components 1 and 2 (C1, C2) can bepurification components, e.g., His₆ (SEQ ID NO: 10), FLAG epitope, orattachment components, e.g., SpyTag.

FIGS. 8A and 8B show SDS-PAGE gels demonstrating the loss ofoligomerization of variant monomers (N17K) having breaking mutations atoligomerization domains as indicated. Reference is made to Example 6.FIGS. 8A and 8B disclose “His6-GSGG” as SEQ ID NO: 11.

FIG. 9 shows a gel of αHL monomers with breaking mutations as indicated,and demonstrating that wild-type αHL monomer (Nemo M) does not enableoligomerization of the mutated monomers. Reference is made to Example 6.

FIGS. 10A-10C show a chromatogram of a SEC purification of a subunitconcatemer of two αHL monomers (10A) and SDS-PAGE gel (10B) of αHLconcatemer of two monomers linked by (GS)₅ (SEQ ID NO:9), tagged at theN-terminus with His6-SpyTag (“His6” disclosed as SEQ ID NO: 10), andexpressed with signal sequence peIB (10C). Reference is made to Example7.

FIG. 11 shows an image of an SDS-PAGE gel that demonstrates that theconcatemer of two monomers shown in FIG. 11 can oligomerize as seen asthe high molecular weight bands in lane 5. Reference is made to Example7.

FIG. 12 shows an image of an SDS-PAGE gel demonstrating thatconcatenated subunits of three and four linked monomers can be expressedand purified. Reference is made to Example 8.

FIG. 13 shows an image of an SDS-PAGE gel that demonstratesoligomerization of αHL subunits having cognate mutations H35I and Y101H,which enable oligomerization of the mutated subunits. Reference is madeto Example 10.

FIG. 14 shows an image of an SDS-PAGE gel that demonstrates thetemperature-dependent oligomerization of monomers that have the H35Gmutation. Reference is made to Example 11.

DETAILED DESCRIPTION

The invention will now be described in detail by way of reference onlyusing the following definitions and examples. All patents andpublications, including all sequences disclosed within such patents andpublications, referred to herein are expressly incorporated byreference.

Unless defined otherwise herein, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which this invention belongs. Singleton, et al.,DICTIONARY OF MICROBIOLOGY AND MOLECULAR BIOLOGY, 2D ED., John Wiley andSons, New York (1994), and Hale & Marham, THE HARPER COLLINS DICTIONARYOF BIOLOGY, Harper Perennial, NY (1991) provide one of skill with ageneral dictionary of many of the terms used in this invention. Althoughany methods and materials similar or equivalent to those describedherein can be used in the practice or testing of the present invention,the preferred methods and materials are described. Practitioners areparticularly directed to Sambrook et al., 1989, and Ausubel F M et al.,1993, for definitions and terms of the art. It is to be understood thatthis invention is not limited to the particular methodology, protocols,and reagents described, as these may vary.

Numeric ranges are inclusive of the numbers defining the range. The term“about” is used herein to mean plus or minus ten percent (10%) of avalue. For example, “about 100” refers to any number between 90 and 110.

Unless otherwise indicated, nucleic acids are written left to right in5′ to 3′ orientation; amino acid sequences are written left to right inamino to carboxy orientation, respectively.

The headings provided herein are not limitations of the various aspectsor embodiments of the invention which can be had by reference to thespecification as a whole. Accordingly, the terms defined immediatelybelow are more fully defined by reference to the specification as awhole.

Definitions

Alpha-hemolysin: As used herein, “alpha-hemolysin,” “α-hemolysin,”“aHL,” “αHL,” “a-HL” and “α-HL” are used interchangeably and refer to aprotein that self-assembles into a heptameric water-filled transmembranechannel from monomers, concatemers of monomers, or a combination ofmonomers and concatemers of monomers.

Amino acid: As used herein, the term “amino acid,” in its broadestsense, refers to any compound and/or substance that can be incorporatedinto a polypeptide chain. In some embodiments, an amino acid has thegeneral structure H₂N—C(H)(R)—COOH. In some embodiments, an amino acidis a naturally-occurring amino acid. In some embodiments, an amino acidis a synthetic amino acid; in some embodiments, an amino acid is aD-amino acid; in some embodiments, an amino acid is an L-amino acid.“Standard amino acid” refers to any of the twenty standard L-amino acidscommonly found in naturally occurring peptides. “Nonstandard amino acid”refers to any amino acid, other than the standard amino acids,regardless of whether it is prepared synthetically or obtained from anatural source. As used herein, “synthetic amino acid” or “non-naturalamino acid” encompasses chemically modified amino acids, including butnot limited to salts, amino acid derivatives (such as amides), and/orsubstitutions. Amino acids, including carboxy- and/or amino-terminalamino acids in peptides, can be modified by methylation, amidation,acetylation, and/or substitution with other chemicals or chemical groupswithout adversely affecting their activity. Amino acids may participatein a disulfide bond. The term “amino acid” is used interchangeably with“amino acid residue,” and may refer to a free amino acid and/or to anamino acid residue of a peptide. It will be apparent from the context inwhich the term is used whether it refers to a free amino acid or aresidue of a peptide. It should be noted that all amino acid residuesequences are represented herein by formulae whose left and rightorientation is in the conventional direction of amino-terminus tocarboxy-terminus.

Base Pair (bp): As used herein, base pair refers to a partnership ofadenine (A) with thymine (T), or of cytosine (C) with guanine (G) in adouble stranded DNA molecule.

Complementary: As used herein, the term “complementary” refers to thebroad concept of sequence complementarity between regions of twopolynucleotide strands or between two nucleotides through base-pairing.It is known that an adenine nucleotide is capable of forming specifichydrogen bonds (“base pairing”) with a nucleotide which is thymine oruracil. Similarly, it is known that a cytosine nucleotide is capable ofbase pairing with a guanine nucleotide.

Expression cassette: An “expression cassette” or “expression vector” isa nucleic acid construct generated recombinantly or synthetically, witha series of specified nucleic acid elements that permit transcription ofa particular nucleic acid in a target cell. The recombinant expressioncassette can be incorporated into a plasmid, chromosome, mitochondrialDNA, plastid DNA, virus, or nucleic acid fragment. Typically, therecombinant expression cassette portion of an expression vectorincludes, among other sequences, a nucleic acid sequence to betranscribed and a promoter.

Heterologous: A “heterologous” nucleic acid construct or sequence has aportion of the sequence which is not native to the cell in which it isexpressed. Heterologous, with respect to a control sequence refers to acontrol sequence (i.e., promoter or enhancer) that does not function innature to regulate the same gene the expression of which it is currentlyregulating. Generally, heterologous nucleic acid sequences are notendogenous to the cell or part of the genome in which they are present,and have been added to the cell, by infection, transfection,transformation, microinjection, electroporation, or the like. A“heterologous” nucleic acid construct may contain a control sequence/DNAcoding sequence combination that is the same as, or different from acontrol sequence/DNA coding sequence combination found in the nativecell.

Host cell: By the term “host cell” is meant a cell that contains avector and supports the replication, and/or transcription ortranscription and translation (expression) of the expression construct.Host cells for use in the present invention can be prokaryotic cells,such as E. coli or Bacillus subtilus, or eukaryotic cells such as yeast,plant, insect, amphibian, or mammalian cells. In general, host cells areprokaryotic, e.g., E. coli.

Isolated: An “isolated” molecule is a nucleic acid molecule that isseparated from at least one other molecule with which it is ordinarilyassociated, for example, in its natural environment. An isolated nucleicacid molecule includes a nucleic acid molecule contained in cells thatordinarily express the nucleic acid molecule, but the nucleic acidmolecule is present extrachromasomally or at a chromosomal location thatis different from its natural chromosomal location.

Modified alpha-hemolysin: As used herein, the term “modifiedalpha-hemolysin” refers to an alpha-hemolysin originated from another(i.e., parental) alpha-hemolysin and contains one or more amino acidalterations (e.g., amino acid substitution, deletion, and/or insertion)compared to the parental alpha-hemolysin. In some embodiments, amodified alpha-hemolysin of the invention is originated or modified froma naturally-occurring or wild-type alpha-hemolysin. In some embodiments,a modified alpha-hemolysin of the invention is originated or modifiedfrom a recombinant or engineered alpha-hemolysin including, but notlimited to, chimeric alpha-hemolysin, fusion alpha-hemolysin or anothermodified alpha-hemolysin. Typically, a modified alpha-hemolysin has atleast one changed phenotype compared to the parental alpha-hemolysin.

Mutation: As used herein, the term “mutation” refers to a changeintroduced into a parental sequence, including, but not limited to,substitutions, insertions, deletions (including truncations). Theconsequences of a mutation include, but are not limited to, the creationof a new character, property, function, phenotype or trait not found inthe protein encoded by the parental sequence.

Nanopore: The term “nanopore,” or “pore” as used herein, generallyrefers to a channel or passage formed or otherwise provided in amembrane. A membrane may be an organic membrane, such as a lipidbilayer, or a synthetic membrane, such as a membrane formed of apolymeric material. The membrane may be a polymeric material. Thenanopore may be disposed adjacent or in proximity to a sensing circuitor an electrode coupled to a sensing circuit, such as, for example, acomplementary metal-oxide semiconductor (CMOS) or field effecttransistor (FET) circuit. In some examples, a nanopore has acharacteristic width or diameter on the order of 0.1 nanometers (nm) toabout 1000 nm. Some nanopores are proteins. Alpha-hemolysin is anexample of a protein nanopore.

Nucleic Acid Molecule: The term “nucleic acid molecule” or “nucleicacid” or “polynucleotide” includes RNA, DNA and cDNA molecules. It willbe understood that, as a result of the degeneracy of the genetic code, amultitude of nucleotide sequences encoding a given protein such asalpha-hemolysin and/or variants thereof may be produced. The presentinvention contemplates every possible variant nucleotide sequence,encoding variant alpha-hemolysin, all of which are possible given thedegeneracy of the genetic code.

Promoter: As used herein, the term “promoter” refers to a nucleic acidsequence that functions to direct transcription of a downstream gene.The promoter will generally be appropriate to the host cell in which thetarget gene is being expressed. The promoter together with othertranscriptional and translational regulatory nucleic acid sequences(also termed “control sequences”) are necessary to express a given gene.In general, the transcriptional and translational regulatory sequencesinclude, but are not limited to, promoter sequences, ribosomal bindingsites, transcriptional start and stop sequences, translational start andstop sequences, and enhancer or activator sequences.

Purified: As used herein, “purified” means that a molecule is present ina sample at a concentration of at least 95% by weight, at least 98%, atleast 99%, or at least 99.5% by weight of the sample in which it iscontained.

Purifying: As used herein, the term “purifying” generally refers tosubjecting transgenic nucleic acid or protein containing cells orextracts thereof to biochemical purification and/or columnchromatography.

Tag: As used herein, the term “tag” refers to a detectable moiety thatmay be atoms or molecules, or a collection of atoms or molecules. A tagmay provide a signature, e.g., an optical, electrochemical, magnetic, orelectrostatic (e.g., inductive, capacitive) signature, which signaturemay be detected with the aid of a nanopore. Typically, when a nucleotideis attached to the tag it is called a “Tagged Nucleotide.” The tag maybe attached to the nucleotide, e.g., via the phosphate moiety.

Time-To-Thread: The term “time to thread” or “TTT” means the time ittakes the polymerase-tag complex to thread the tag into the barrel ofthe nanopore.

Variant: As used herein, the term “variant” refers to a modified proteinwhich displays altered characteristics when compared to the parentalprotein, e.g., altered ionic conductance, altered time to thread, etc.

Variant hemolysin: The term “variant hemolysin gene” or “varianthemolysin” means, respectively, that the nucleic acid sequence of thealpha-hemolysin gene from Staphylococcus aureus has been altered byremoving, adding, and/or manipulating the coding sequence or the aminoacid sequence or the expressed protein has been modified consistent withthe invention described herein.

Vector: As used herein, the term “vector” refers to a nucleic acidconstruct designed for transfer between different host cells. An“expression vector” refers to a vector that has the ability toincorporate and express heterologous DNA fragments in a foreign cell.Many prokaryotic and eukaryotic expression vectors are commerciallyavailable. Selection of appropriate expression vectors is within theknowledge of those having skill in the art.

Wild-type: As used herein, the term “wild-type” refers to a gene or geneproduct which has the sequence and/or characteristics of that gene orgene product when isolated from a naturally-occurring source.

Percent homology: The term “% homology” is used interchangeably hereinwith the term “% identity” herein and refers to the level of nucleicacid or amino acid sequence identity between the nucleic acid sequencethat encodes any one of the inventive polypeptides or the inventivepolypeptide's amino acid sequence, when aligned using a sequencealignment program.

For example, as used herein, 80% homology means the same thing as 80%sequence identity determined by a defined algorithm, and accordingly ahomologue of a given sequence has greater than 80% sequence identityover a length of the given sequence. Exemplary levels of sequenceidentity include, but are not limited to, 80, 85, 90, 95, 98% or moresequence identity to a given sequence, e.g., the coding sequence for anyone of the inventive polypeptides, as described herein.

Exemplary computer programs which can be used to determine identitybetween two sequences include, but are not limited to, the suite ofBLAST programs, e.g., BLASTN, BLASTX, and TBLASTX, BLASTP and TBLASTN,publicly available on the Internet. See also, Altschul, et al., 1990 andAltschul, et al., 1997.

Sequence searches are typically carried out using the BLASTN programwhen evaluating a given nucleic acid sequence relative to nucleic acidsequences in the GenBank DNA Sequences and other public databases. TheBLASTX program is preferred for searching nucleic acid sequences thathave been translated in all reading frames against amino acid sequencesin the GenBank Protein Sequences and other public databases. Both BLASTNand BLASTX are run using default parameters of an open gap penalty of11.0, and an extended gap penalty of 1.0, and utilize the BLOSUM-62matrix. (See, e.g., Altschul, S. F., et al., Nucleic Acids Res.25:3389-3402, 1997.)

A preferred alignment of selected sequences in order to determine “%identity” between two or more sequences, is performed using for example,the CLUSTAL-W program in MacVector version 13.0.7, operated with defaultparameters, including an open gap penalty of 10.0, an extended gappenalty of 0.1, and a BLOSUM 30 similarity matrix.

“Oligomeric proteins”: The term “oligomeric proteins” herein refer toproteins that can be composed of multiple identical subunits, multipledistinct subunits, or a mixture of identical and distinct subunits.Proteins with identical subunits are termed “homo-oligomers”. Proteinscontaining two or more distinct polypeptide subunits are termed“hetero-oligomers”.

“Hetero-heptameric protein”: The term “hetero-heptameric protein” hereinrefers to a protein containing two or more distinct subunitpolypeptides, wherein each polypeptide comprises one or more αHLmonomers that form a protein of seven monomers.

“Oligomerization subunit”: The term “oligomerization subunit” or“subunit” herein refer to a polypeptide that comprises an amino acidsequence for at least one αHL monomer, or for at least one αHLconcatemer of two, three, four, five, six, or seven monomers that arelinked to each other by a linker, and are each encoded by a singlepolynucleotide.

“Oligomerization domain”: The term “oligomerization domain” hereinrefers to amino acids in a region of one subunit that can interact withamino acids in a region of another subunit to enable oligomerization ofthe subunits. Each monomer subunit or each concatemer subunit ofmonomers has first and second oligomerization domains.

“Breaking mutation”: The term “breaking mutation” herein refers to amutation in an αHL subunit that does not allow for inter-subunitinteraction with a wild-type αHL subunit thereby inhibitingoligomerization.

“Rescue mutation”: The term “rescue mutation” herein refers to amutation that is not a breaking mutation, which when present on theoligomerization domain of a first subunit can interface with a breakingmutation in the oligomerization domain of a second subunit to enableinter-subunit interaction thereby allowing oligomerization of thesubunits. Rescue mutations can also enable oligomerization with wildtype subunits. “Rescue mutations” can also be referred to as“compensating mutations”.

“Cognate mutation”: the term “cognate mutation” herein refers to abreaking mutation on the oligomerization domain of a first subunit thatcan interface with a breaking mutation in the oligomerization domain ofa second subunit to enable inter-subunit interaction thereby allowingoligomerization of the subunits.

“Self-rescue mutation”: the term “self-rescue mutation” herein refers toa mutation that is a breaking mutation at a first temperature (e.g.,room temperature) and converts to a cognate mutation at a secondtemperature (e.g., 37° C.). It is understood that the first temperaturemay be either higher or lower than the second temperature.

“Mutated variant”: The term “mutated variant” herein refers to a variantαHL subunit, e.g., monomer, that has been further modified to introduceone or more mutations, e.g., substitutions, in one or both of theoligomerization domains of an αHL subunit.

“Oligomerization mutant”: the term “oligomerization mutant” hereinrefers to an αHL subunit having one or more mutations in one or botholigomerization domains.

Nomenclature

In the present description and claims, the conventional one-letter andthree-letter codes for amino acid residues are used.

For ease of reference, variants of the application are described by useof the following nomenclature:

Original amino acid(s): position(s): substituted amino acid(s).According to this nomenclature, for instance the substitution ofthreonine by an arginine in position 17 is shown as:

-   -   Thr17Arg or T17R

Multiple mutations are separated by plus signs, i.e.:

-   -   Thr17Arg+Glu34Ser or T17R+E34S        representing mutations in positions 17 and 34 substituting        arginine and serine for threonine and glutamic acid,        respectively.

When one or more alternative amino acid residues may be inserted in agiven position it is indicated as: T17R/K, or T17R or T17K.

Site-Directed Mutagenesis of Alpha-Hemolysin

Staphylococcus aureus alpha hemolysin wild type sequences are providedherein (SEQ ID NO:1, nucleic acid coding region; SEQ ID NO:3, proteincoding region) and available elsewhere (National Center forBioinformatics or GenBank Accession Numbers M90536 and AAA26598).

Point mutations may be introduced using QuikChange Lightning 2 kit(Stategene/Agilent) following manufacturer's instructions.

Primers can be ordered from commercial companies, e.g., IDT DNA.

Nanopore Assembly and Insertion

The methods described herein can use a nanopore having a polymeraseattached to the nanopore. In some cases, it is desirable to have one andonly one polymerase per nanopore (e.g., so that only one nucleic acidmolecule is sequenced at each nanopore). However, many nanopores,including alpha-hemolysin (αHL), can be multimeric proteins having aplurality of subunits (e.g., 7 subunits for αHL). The subunits can beidentical copies of the same polypeptide. Provided herein are multimericproteins (e.g., nanopores) having a defined ratio of modified subunits(e.g., a-HL variants) to un-modified subunits (e.g., a-HL). Alsoprovided herein are methods for producing multimeric proteins (e.g.,nanopores) having a defined ratio of modified subunits to un-modifiedsubunits.

With reference to FIG. 27 of WO2014/074727, a method for assembling aprotein having a plurality of subunits comprises providing a pluralityof first subunits 2705 and providing a plurality of second subunits2710, where the second subunits are modified when compared with thefirst subunits. In some cases, the first subunits are wild-type (e.g.,purified from native sources or produced recombinantly). The secondsubunits can be modified in any suitable way. In some cases, the secondsubunits have a protein (e.g., a polymerase) attached (e.g., as a fusionprotein).

The modified subunits can comprise a chemically reactive moiety (e.g.,an azide or an alkyne group suitable for forming a linkage). In somecases, the method further comprises performing a reaction (e.g., a Clickchemistry cycloaddition) to attach an entity (e.g., a polymerase) to thechemically reactive moiety.

The method can further comprise contacting the first subunits with thesecond subunits 2715 in a first ratio to form a plurality of proteins2720 having the first subunits and the second subunits. For example, onepart modified αHL subunits having a reactive group suitable forattaching a polymerase can be mixed with six parts wild-type αHLsubunits (i.e., with the first ratio being 1:6). The plurality ofproteins can have a plurality of ratios of the first subunits to thesecond subunits. For example, the mixed subunits can form severalnanopores having a distribution of stoichiometries of modified toun-modified subunits (e.g., 1:6, 2:5, 3:4).

In some cases, the proteins are formed by simply mixing the subunits. Inthe case of αHL nanopores for example, a detergent (e.g., deoxycholicacid) can trigger the αHL monomer to adopt the pore conformation. Thenanopores can also be formed using a lipid (e.g.,1,2-diphytanoyl-sn-glycero-3-phosphocholine (DPhPC) or1,2-di-0-phytanyl-s/n-glycero-3-phosphocholine (DoPhPC)) and moderatetemperature (e.g., less than about 100° C.). In some cases, mixing DPhPCwith a buffer solution creates large multi-lamellar vesicles (LMV), andadding αHL subunits to this solution and incubating the mixture at 40°C. for 30 minutes results in pore formation.

If two different types of subunits are used (e.g., the natural wild typeprotein and a second αHL monomer which can contain a single pointmutation), the resulting proteins can have a mixed stoichiometry (e.g.,of the wild type and mutant proteins). The stoichiometry of theseproteins can follow a formula which is dependent upon the ratio of theconcentrations of the two proteins used in the pore forming reaction.This formula is as follows:

100 P _(m)=100[n!/m!(n−m)!]·f _(mut) ^(m) ·f _(wt) ^(n˜m), where

-   -   P_(m)=probability of a pore having m number of mutant subunits    -   n=total number of subunits (e.g., 7 for αHL)    -   m=number of “mutant” subunits    -   f_(mut)=fraction or ratio of mutant subunits mixed together    -   f_(wt)=fraction or ratio of wild-type subunits mixed together

The method can further comprise fractionating the plurality of proteinsto enrich proteins that have a second ratio of the first subunits to thesecond subunits 2725. For example, nanopore proteins can be isolatedthat have one and only one modified subunit (e.g., a second ratio of1:6). However, any second ratio is suitable. A distribution of secondratios can also be fractionated such as enriching proteins that haveeither one or two modified subunits. The total number of subunitsforming the protein is not always 7 (e.g., a different nanopore can beused or an alpha-hemolysin nanopore can form having six subunits) asdepicted in FIG. 27 of WO2014/074727. In some cases, proteins havingonly one modified subunit are enriched. In such cases, the second ratiois 1 second subunit per (n−1) first subunits where n is the number ofsubunits comprising the protein.

The first ratio can be the same as the second ratio, however this is notrequired. In some cases, proteins having mutated monomers can form lessefficiently than those not having mutated subunits. If this is the case,the first ratio can be greater than the second ratio (e.g., if a secondratio of 1 mutated to 6 non-mutated subunits are desired in a nanopore,forming a suitable number of 1:6 proteins may require mixing thesubunits at a ratio greater than 1:6). Conversely, if mutated monomersare able to oligomerize more efficiently, then the first ratio can beless than the second ratio (e.g., if a second ratio of 1 mutated to 6non-mutated monomers are desired in a nanopore, forming a suitablenumber of 1:6 proteins may require mixing the subunits at a ratio lessthan 1:6).

Proteins having different second ratios of subunits can behavedifferently (e.g., have different retention times) in a separation. Insome cases, the proteins are fractionated using chromatography, such asion exchange chromatography or affinity chromatography. Since the firstand second subunits can be identical apart from the modification, thenumber of modifications on the protein can serve as a basis forseparation. In some cases, either the first or second subunits have apurification tag (e.g., in addition to the modification) to allow orimprove the efficiency of the fractionation. In some cases, apoly-histidine tag (His-tag), a streptavidin tag (Strep-tag), or otherpeptide tag is used. In some instances, the first and second subunitseach comprise different tags and the fractionation step fractionates onthe basis of each tag. In the case of a His-tag, a charge is created onthe tag at low pH (Histidine residues become positively charged belowthe pKa of the side chain). With a significant difference in charge onone of the αHL molecules compared to the others, ion exchangechromatography can be used to separate the oligomers which have 0, 1, 2,3, 4, 5, 6, or 7 of the “charge-tagged” αHL subunits. In principle, thischarge tag can be a string of any amino acids which carry a charge,e.g., a uniform charge. FIG. 28 and FIG. 29 of WO2014/074727 showexamples of fractionation of nanopores based on a His-tag. FIG. 28 showsa plot of ultraviolet absorbance at 280 nanometers, ultravioletabsorbance at 260 nanometers, and conductivity. The peaks correspond tonanopores with various ratios of modified and unmodified subunits. FIG.29 of WO2014/074727 shows fractionation of αHL nanopores and mutantsthereof using both His-tag and Strep-tags.

In some cases, an entity (e.g., a polymerase) is attached to the proteinfollowing fractionation. The protein can be a nanopore and the entitycan be a polymerase. In some instances, the method further comprisesinserting the proteins having the second ratio subunits into a bilayer.

In some situations, a nanopore can comprise a plurality of subunits. Apolymerase can be attached to one of the subunits and at least one andless than all of the subunits comprise a first purification tag. In someexamples, the nanopore is alpha-hemolysin or a variant thereof. In someinstances, all of the subunits comprise a first purification tag or asecond purification tag. The first purification tag can be apoly-histidine tag (e.g., on the subunit having the polymeraseattached).

Polymerase Attached to Nanopore

In some cases, a polymerase (e.g., DNA polymerase) is attached to and/oris located in proximity to the nanopore. The polymerase can be attachedto the nanopore before or after the nanopore is incorporated into themembrane. In some instances, the nanopore and polymerase are a fusionprotein (i.e., single polypeptide chain).

The polymerase can be attached to the nanopore in any suitable way. Insome cases, the polymerase is attached to the nanopore (e.g., hemolysin)protein monomer and then the full nanopore heptamer is assembled (e.g.,in a ratio of one monomer with an attached polymerase to 6 nanopore(e.g., hemolysin) monomers without an attached polymerase). The nanoporeheptamer can then be inserted into the membrane.

Another method for attaching a polymerase to a nanopore involvesattaching a linker molecule to a hemolysin monomer or mutating ahemolysin monomer to have an attachment site and then assembling thefull nanopore heptamer (e.g., at a ratio of one monomer with linkerand/or attachment site to 6 hemolysin monomers with no linker and/orattachment site). A polymerase can also be attached to a concatemer ofαHL monomers. For example, FIGS. 7A-7F show that concatemers of two ormore αHL monomers can comprise attachment components to which an enzyme,e.g., a polymerase, can be linked. Accordingly, a polymerase can belinked to a concatemer of two or more monomers, which can beoligomerized with other concatemers and/or monomers to provide ananopore, e.g., a heptameric αHL nanopore, comprising a polymeraseenzyme. A second polymerase can also be linked to a monomer or to aconcatemer of monomers. A polymerase can then be attached to theattachment site or attachment linker (e.g., in bulk, before insertinginto the membrane). The polymerase can also be attached to theattachment site or attachment linker after the (e.g., heptamer) nanoporeis formed in the membrane. In some cases, a plurality ofnanopore-polymerase pairs is inserted into a plurality of membranes(e.g., disposed over the wells and/or electrodes) of the biochip. Insome instances, the attachment of the polymerase to the nanopore complexoccurs on the biochip above each electrode.

The polymerase can be attached to the nanopore with any suitablechemistry (e.g., covalent bond and/or linker). In some cases, thepolymerase is attached to the nanopore with molecular staples. In someinstances, molecular staples comprise three amino acid sequences(denoted linkers A, B and C). Linker A can extend from a hemolysinmonomer, Linker B can extend from the polymerase, and Linker C then canbind Linkers A and B (e.g., by wrapping around both Linkers A and B) andthus the polymerase to the nanopore. Linker C can also be constructed tobe part of Linker A or Linker B, thus reducing the number of linkermolecules.

In some instances, the polymerase is linked to the nanopore usingSolulink™ chemistry. Solulink™ can be a reaction between HyNic(6-hydrazino-nicotinic acid, an aromatic hydrazine) and 4FB(4-formylbenzoate, an aromatic aldehyde). In some instances, thepolymerase is linked to the nanopore using Click chemistry (availablefrom LifeTechnologies for example). In some cases, zinc finger mutationsare introduced into the hemolysin molecule and then a molecule is used(e.g., a DNA intermediate molecule) to link the polymerase to the zincfinger sites on the hemolysin.

Stoichiometry and Arrangement of αHL Subunits into Heptameric Pores

In another aspect, hetero-oligomeric αHL heptamers and methods forpreparing the heptamers are provided. The hetero-oligomeric heptamerscan be formed by regulating the stoichiometry and the sequentialarrangement of their subunit components. The sequential arrangement isdetermined by the interaction of mutations in oligomerization domains ofthe subunits.

Heptameric wild-type αHL pores are formed by self-assembly of sevenwild-type monomer subunits. Each monomer subunit comprises a first and asecond oligomerization domain whereby the first oligomerization domainof one subunit interacts with the second oligomerization domain ofanother subunit to enable the self-assembly of monomeric subunits into aheptameric αHL pore. The first oligomerization domain region, i.e., site1 of each monomer subunit, comprises amino acids corresponding to aminoacid positions 20-28, 35-42, and 53-61 of αHL of SEQ ID NO:3. The secondinterface region, i.e., site 2 of each αHL monomer, comprises aminoacids corresponding to amino acid positions 158-164, 95-104, 43 to 48,and 228 to 236 of the αHL subunit of SEQ ID NO:3. FIG. 6A illustratesthe positioning of the first (601) and second (602) oligomerizationdomain, respectively, of a monomer subunit. The positioning of theinteraction of the oligomerization domains of the seven monomer subunitsnumbered i, ii, iii, iv, v, vi, and vii as they are oligomerized into aheptameric αHL pore is shown schematically in FIG. 6B.

However, assembly of engineered monomers can give rise to oligomershaving undesirable stoichiometries. For example, assembly of engineeredsubunits can give rise to octamers consisting of four dimer subunits, orto hexamers of three dimer subunits (Hammerstein et al., 2011).Therefore, it would be advantageous to control the assembly of the αHLsubunits to provide the heptameric form, which is the stoichiometry thatenables the sensing capability of an αHL pore.

In one embodiment, the oligomerization of αHL subunits into a heptamericpore can be obtained by introducing mutations at interfaces, i.e.,oligomerization domains, on each of the subunits to disrupt thesubunit-subunit interaction. As described above, and depicted in FIGS.6A and 6B, there are two oligomerization domains on each subunit, andone or more mutations can be introduced in the first and/or the secondoligomerization domain of each subunit to inhibit inter-subunit contactand thereby prevent oligomerization of subunits into αHL multimershaving undesirable subunit stoichiometries. Mutations that inhibitinter-subunit interactions are herein referred to as breaking mutations.Each oligomerization domain of each subunit can be modified to compriseone or more breaking mutations. In some embodiments, one or more aminoacids in the first oligomerization domain can be mutated to provide oneor more breaking mutations. Similarly, one or more amino acids in thesecond oligomerization domain can be mutated to provided one or morebreaking mutations. In other embodiments, one or more amino acids in thefirst and in the second oligomerization domain of each subunit can bemutated to provide one or more breaking mutation.

Mutations that can be made at oligomerization domains includesubstitutions, deletions, and insertions. Preferred mutations of aminoacids in these domains are amino acid substitutions.

In some embodiments, one or more of the amino acids corresponding topositions 20-28, 35-42, and 53-61 of the first oligomerization domain ofthe αHL subunit of SEQ ID NO:3 are mutated to introduce one or morebreaking mutations into the first oligomerization domain of an αHLsubunit. In some embodiments, breaking mutations at the firstoligomerization domain include D24A, V26D, K375, H35I, H35D, H35E, H35Land D24A+V26D+K37S of SEQ ID NO:3.

In other embodiments, one or more of the amino acids corresponding topositions 158-164, 95-104, 43 to 48, and 228 to 236 of the αHL subunitof SEQ ID NO:3 are mutated to introduce one or more breaking mutationsinto the second oligomerization domain of an αHL subunit. In someembodiments, breaking mutations at the second oligomerization domaininclude T233R, S99K, Y101D, Y101H, and T233R+S99K of SEQ ID NO:3. FIGS.8A and 8B show that in the presence of lipid, mutated monomers havingbreaking mutations at the first and/or second oligomerization domainscannot be reconstituted into an oligomeric protein.

To enable controlled oligomerization of subunits comprising breakingmutations, rescue and/or cognate mutations are introduced in one or bothof the oligomerization domains of subunits to revert the effect of thebreaking mutations and allow for the requisite inter-subunit interactionthat is necessary for oligomerization and formation of the heptamericnanopore. FIG. 6C shows the types of interactions between mutations onoligomerization domains of αHL subunits that allow or inhibitoligomerization. Breaking mutations 1 and 2 (BM1, BM2) inhibitoligomerization of the subunits via the domains in which they reside.Similarly, a breaking mutation, e.g., BM1, inhibits oligomerization witha wild-type subunit. Inhibition of oligomerization is shown by thecrossed broken arrow lines. Reversal of the breaking effect of breakingmutations, e.g., BM1, is enabled by cognate mutations (CM) and/or rescuemutations (RM). Enablement of oligomerization is depicted by the solidarrow lines.

Cognate mutations (CM), which are themselves breaking mutations, whenpresent on the oligomerization domain of a first subunit, can interactwith breaking mutations (e.g., BM1) on the oligomerization domain ofanother subunit to restore the ability of the two subunits tooligomerize. The pairing of the breaking and cognate mutations alsospecifies which two subunits can interact in the process. In someembodiments, a breaking mutation on a first oligomerization domain ofone subunit, i.e., a preceding subunit, allows for the interaction of asecond subunit via a cognate mutation on a second oligomerization domainon a second subunit, i.e., a following subunit, as the cognate mutationreverts the effect of the breaking mutation to allow inter-subunitinteraction via the first and second domains.

Rescue mutations (RM), which are not breaking mutations, when present onthe oligomerization domain of a first subunit, can interact withbreaking mutations (e.g., BM1) on the oligomerization domain of anothersubunit to restore the ability of the two subunits to oligomerize. Thepairing of the breaking and rescue mutations also specifies which twosubunits can interact in the process. In some embodiments, a breakingmutation on a first oligomerization domain of one subunit, i.e., apreceding subunit, allows for the interaction of a second subunit via arescue mutation on a second oligomerization domain on a second subunit,i.e., a following subunit, as the rescue mutation reverts the effect ofthe breaking mutation to allow inter-subunit interaction via the firstand second domains.

Thus, in some embodiments, at least one of the mutations of the one ormore of the amino acids corresponding to positions 20-28, 35-42, and53-61 of the first oligomerization domain of the αHL subunit of SEQ IDNO:3 is a breaking mutation, and/or at least one of the mutations of theone or more of the amino acids corresponding to positions 158-164,95-104, 43 to 48, and 228 to 236 of the second oligomerization domain ofthe αHL subunit of SEQ ID NO:3 is a rescue and/or cognate mutation thatenables inter-subunit interaction with corresponding second and firstoligomerization domains of following or preceding subunits,respectively. Alternatively, at least one of the mutations of the one ormore of the amino acids corresponding to positions 158-164, 95-104, 43to 48, and 228 to 236 of the second oligomerization domain of SEQ IDNO:3 is a breaking mutation, and/or at least one of the mutations one ormore of the amino acids corresponding to positions 20-28, 35-42, and53-61 of the first oligomerization domain αHL subunit of SEQ ID NO:3 isa cognate and/or rescue mutation. It is understood that breakingmutations in one domain of one subunit can interact with rescue and/orcognate mutations of a domain of another subunit to enableoligomerization of the subunits. The one or more breaking mutationsinhibit oligomerization of the subunit by at least about 10%, 20%, 30%40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, or more when compared to theoligomerization of the unmutated αHL subunits. The one or more rescueand/or cognate mutations on one subunit enable inter-subunit interactionwith breaking mutations on another subunit to obtain at least about 10%,20%, 30% 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, or more oligomerizationof the subunits when compared to the oligomerization of the unmutatedαHL subunits or when compared to the oligomerization of the subunitwithout the rescue and/or cognate mutation with the subunit thatcomprises the breaking mutation.

In another embodiment, inter-subunit interaction can be establishedbetween a breaking mutation in a first oligomerization domain on apreceding subunit and a rescue mutation and/or a cognate mutation in thesecond domain of a following subunit. Accordingly, at least one breakingmutation can be made in each of the first oligomerization domain of apreceding subunit and at least one breaking mutation can be made in eachof the second oligomerization domain of a following subunit. In someembodiments, at least one of the breaking mutations in the secondoligomerization domain of a following subunit is a cognate mutation thatwhen paired with a breaking mutation on the preceding subunit allows forinter-subunit interaction, thereby enabling oligomerization of thesubunits. In other embodiments, the mutation in the secondoligomerization domain of a following subunit is a rescue mutation thatwhen paired with a breaking mutation on the first oligomerization domainof a preceding subunit, allows for inter-subunit interaction, therebyenabling oligomerization of the subunits.

In some embodiments, oligomerization subunits can comprise at least onebreaking mutation that can convert itself to a cognate mutation, i.e.,the breaking mutation can be a self-rescue mutation. Applicantsdiscovered that there exist breaking mutations that inhibitoligomerization of monomers at temperatures below 30° C., but whichenable oligomerization when the process is performed at temperaturesgreater than 30° C. For example, and in reference to Example 11 and FIG.14, monomers having a breaking mutation H35G of SEQ ID NO:3, are unableto oligomerize at 25° C. However, when reconstitution of the samemonomers, i.e., monomers having the same H35G mutation, the monomers areable to oligomerize at 37° C. Accordingly, in some embodiments,heptamers can comprise at least one breaking mutation in the firstand/or second oligomerization domain that is a self-rescue mutation,which enables oligomerization of subunits. In some embodiments,conversion of breaking mutations to self-rescue mutation takes place attemperatures between 30° C. and 50° C., between 35° C. and 45° C., orbetween 37° C. and 43° C. In some embodiments, conversion of breakingmutations to self-rescue mutation takes place at any of about 30° C.,35° C., 40° C., 45° C., or 50° C. It is understood that absentdenaturation, conversion of breaking mutations to self-rescue mutationcan take place at temperatures greater than 50° C. In some embodiments,the self-rescue mutation is the amino acid substitution corresponding toH35G in SEQ ID NO:3.

Self-rescue mutations are particularly useful as it is advantageous tooligomerize pore subunits, e.g., monomers, at higher temperatures.Protein expression at lower temperatures of variants comprisingself-rescue mutations allows for accurate determination of monomerconcentration due to the inhibition/blockade of oligomerization. Incontrast, wild-type monomers can oligomerize and exist as a mixture ofmonomers and oligomers. The WT oligomerization leads to inaccuratemeasurements of monomer concentration. Knowledge of the trueconcentration of monomers in a solution is critical in obtaining thecorrect ratio of subunit types needed to create the desired heptamericpore. Accordingly, a self-rescue mutation, which behaves as a breakingmutation at lower temperatures prevents oligomerization and therebyallows for accurate determination of monomer concentration.Subsequently, the desired heptameric pore can be obtained at highertemperatures at which the same breaking mutation converts to aself-rescue mutation to allow for the desired oligomerization.

Oligomerization subunits can be monomers, or they can be concatemers oftwo linked monomers (dimer concatemer), three linked monomers (trimerconcatemer), four linked monomers (tetramer concatemer), five linkedmonomers (penta-concatemers), six linked monomers (hexa-concatemers),and seven linked monomers (hepta-concatemers). The first oligomerizationdomain of a concatemer subunit is the first oligomerization domain ofthe first monomer of the concatemer (N-terminal); and the secondoligomerization domain of the concatemer subunit is the secondoligomerization domain of the last (C-terminal) monomer of theconcatemer. The monomer subunits can be linked by linker polypeptidesthat join the C-terminal end of a preceding monomer to the N-terminalend of a following monomer. FIG. 6D shows an oligomerization subunitthat is a concatemer of two αHL monomers vii and i, which are joined bya linker (

) In this concatemer subunit, the first oligomerization domain (□) isthe first oligomerization domain of the first subunit vii, and thesecond oligomerization domain of the concatemer subunit (

) is the second oligomerization domain of the second αHL monomer in theconcatemer (i). The linker can be any form of molecule that links thefirst and second regions by covalent forces. In particular, the linkercan be a peptide or polypeptide of any length that will function in thecontext of the invention, including any of about 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 15, 20, 25, 30, 40, 50, or more amino acids, or any number orrange of amino acids that will work for the purposes of the invention.The amino acid linker can include synthetic or naturally occurring aminoacid residues. Those of ordinary skill in the art will be able todetermine and test any number of types and lengths of linkers. Thelinker can be between the C-terminus of the preceding subunit and theN-terminus of the following subunit. In some embodiments, the linker isa flexible linker of up to 5, up to 10, up to 15, up to 20, up to 25, orup to 30 amino acids. In some embodiments the linker is of about 10amino acids. In other embodiments, the linker is of about 5 amino acids.

In some embodiments, an attachment component and/or a purificationcomponent can be provided at one or both the C-terminus and N-terminusof the concatemer polypeptide. Purification components include but arenot limited to His6 (SEQ ID NO: 10) and FLAG epitopes. Attachmentcomponents include but are not limited to the SpyTag/SpyCatcher peptidesystem (Zakeri et al. PNAS109:E690-E697 [2012]), native chemicalligation (Thapa et al., Molecules 19:14461-14483 [2014]), sortase system(Wu and Guo, J Carbohydr Chem 31:48-66 [2012]; Heck et al., ApplMicrobiol Biotechnol 97:461-475 [2013])), transglutaminase systems(Dennler et al., Bioconjug Chem 25:569-578 [2014]), formylglycinelinkage (Rashidian et al., Bioconjug Chem 24:1277-1294 [2013]), or otherchemical ligation techniques known in the art. Attachment components canserve to attach an enzyme, e.g., a polymerase, to the αHL subunit.Enzymes that can be attached to an αHL pore include polymerases, e.g.,DNA polymerases, RNA polymerases, and reverse transcriptases. In someembodiments, a polymerase can be attached to two different αHL subunitswithin an αHL heptameric pore by attachment components on two differentαHL subunits. In other embodiments, a polymerase can be attached tothree different αHL subunits of an αHL heptameric pore by attachmentcomponents on three different αHL subunits. In other embodiments, two ormore enzymes may be attached to any number of αHL subunits. FIGS. 7A-7Falso show examples of concatemer subunits of two, three, and fourmonomers and the positioning of attachment and/or purificationcomponents.

The mutated monomers or concatemers of monomers comprising the one ormore mutations in the first and second oligomerization domains canfurther comprise one or more mutations in regions other than the firstand second oligomerization domain of the polypeptide monomer or monomerconcatemer. For example, the variant αHL monomer polypeptide thatcomprises an amino acid substitution at a position corresponding toposition 12 or 17 of SEQ ID NO:3, which alters the TTT of the αHLrelative to that of a parent αHL, can be further mutated to comprisemutations, e.g., amino acid substitutions at first and secondoligomerization domains to confer the ability of the subunits to form aheptameric αHL pore.

Further embodiments relate to nucleic acids that encode the mutated αHLoligomerization subunits of monomers and of concatemers of monomers.These nucleic acids, in some embodiments, encode oligomerizationsubunits having one or more mutations at a first oligomerization domainand/or at a second oligomerization domain as described elsewhere herein.In some embodiments, the starting ATG of a monomer that is linked to theC-terminus of a preceding monomer in a concatemer subunit is removed toavoid late initiation products.

The polynucleotides can further comprise a signal sequence.

In some embodiments, the polynucleotides further comprise sequence(s)that encode linkers that join monomer units in concatemers of monomers,as described elsewhere herein.

In other embodiments, the polynucleotides comprise sequence(s) thatencode purification and/or attachment components.

In some embodiments, the polynucleotides comprise sequence(s) thatencode a purification component and/or an attachment component (FIGS.7A-7F). The purification and/or attachment components can be positionedat the N-terminus and/or C-terminus of the oligomerization subunit.Purification and/or attachment components can also be attached at anyregion within the polypeptide, i.e., at a region that is between theC-terminus and the N-terminus. In some embodiments, the purification andattachment components can be positioned at the N-terminus or C-terminusof the oligomerization subunit. In some embodiments, at least onepurification component can be positioned at the N-terminus and theattachment component can be positioned at the C-terminus of theoligomerization subunit. In other embodiments, at least one purificationcomponent can be positioned at the C-terminus and the attachmentcomponent can be positioned at the N-terminus of the oligomerizationsubunit. In some embodiments, at least one attachment component can bepositioned within the polypeptide and the purification component can bepositioned at the C-terminus. In some embodiments, at least oneattachment component can be positioned within the polypeptide and thepurification component can be positioned at the N-terminus.

In some aspects of the invention, the nucleic acids are expressible toproduce polypeptides. The polypeptides may be expressed in prokaryoticcells or eukaryotic cells or expressed in a cell free system. Preferredcells for expression include, but are not limited to, bacterial cells,insect cells, yeast cells, and mammalian cells.

Another aspect of the current invention comprises vectors that comprisea nucleic acid encoding all or part of a polypeptide of the presentinvention. The vectors may, for example, be cloning or expressionvectors. The cloning vectors of the invention may be comprised in anysuitable recombinant host cell, as described elsewhere herein or knownto those of skill in the art.

Methods for preparing hetero-oligomeric αHL heptamer proteins are alsoprovided. A polynucleotide encoding a mutated αHL subunit is expressedin a host cell. Different polynucleotides encoding different mutated αHLsubunits are expressed individually in different host cells. Optionally,each of the expressed subunit polypeptides is purified, and subsequentlymixed to allow for oligomerization into a hetero-oligomeric αHLheptamer. In some embodiments, the method can comprise providing a firstpolynucleotide encoding a first mutated αHL subunit having one or morebreaking mutations in the first oligomerization domain and/or in thesecond oligomerization domain, culturing a host cell transformed ortransfected with an expression vector encoding the first polynucleotideencoding the first mutated αHL subunit; providing a secondpolynucleotide encoding a second mutated αHL subunit having at least onerescue and/or cognate mutation in a first and/or second oligomerizationdomain, culturing a second host cell transformed or transfected with anexpression vector encoding the second polynucleotide encoding the secondαHL subunit, wherein the first and second mutated αHL subunitsoligomerize to form at least an αHL dimer, at least an αHL trimer, atleast an HL tetramer, at least an αHL pentamer, at least an αHL hexamer,or at least an αHL heptamer. The method can further comprise purifyingthe first and a second mutated αHL subunits. Purified αHL subunits canbe oligomerized in the presence of lipid to form heptameric αHL pores.It is understood that heptameric αHL pores can be formed byoligomerization of single monomer subunits, e.g., seven αHL monomersubunits, by oligomerization of concatemer subunits, e.g., a concatemerof three αHL monomers and a concatemer of four αHL monomers, or byoligomerization of a mixture of αHL monomer subunits, and αHL concatemersubunits, e.g., three αHL monomer subunits and a concatemer subunit offour αHL monomers. The αHL pores retain the ability to identifynucleotide tags as the nucleotides are incorporated into a newpolynucleotide strand by a polymerase that is attached to the αHL pore.

Apparatus Set-Up

The nanopore may be formed or otherwise embedded in a membrane disposedadjacent to a sensing electrode of a sensing circuit, such as anintegrated circuit. The integrated circuit may be an applicationspecific integrated circuit (ASIC). In some examples, the integratedcircuit is a field effect transistor or a complementary metal-oxidesemiconductor (CMOS). The sensing circuit may be situated in a chip orother device having the nanopore, or off of the chip or device, such asin an off-chip configuration. The semiconductor can be anysemiconductor, including, without limitation, Group IV (e.g., silicon)and Group III-V semiconductors (e.g., gallium arsenide). See, forexample, WO 2013/123450, for the apparatus and device set-up for sensinga nucleotide or tag.

Pore based sensors (e.g., biochips) can be used forelectro-interrogation of single molecules. A pore based sensor caninclude a nanopore of the present disclosure formed in a membrane thatis disposed adjacent or in proximity to a sensing electrode. The sensorcan include a counter electrode. The membrane includes a trans side(i.e., side facing the sensing electrode) and a cis side (i.e., sidefacing the counter electrode).

In the experimental disclosure which follows, the followingabbreviations apply: eq (equivalents); M (Molar); μM (micromolar); N(Normal); mol (moles); mmol (millimoles); μmol (micromoles); nmol(nanomoles); g (grams); mg (milligrams); kg (kilograms); μg(micrograms); L (liters); ml (milliliters); μl (microliters); cm(centimeters); mm (millimeters); μm (micrometers); nm (nanometers); ° C.(degrees Centigrade); h (hours); min (minutes); sec (seconds); msec(milliseconds).

EXAMPLES

The present invention is described in further detail in the followingexamples which are not in any way intended to limit the scope of theinvention as claimed. The attached Figures are meant to be considered asintegral parts of the specification and description of the invention.All references cited are herein specifically incorporated by referencefor all that is described therein. The following examples are offered toillustrate, but not to limit the claimed invention.

Example 1 Expression and Recovery

This example illustrates the expression and recovery of protein frombacterial host cells, e.g., E. coli.

DNA encoding the wild-type a-HL was purchased from a commercial source.The sequence was verified by sequencing.

Plasmid construction. The gene encoding either a wild-type or variantα-hemolysin was inserted into a pPR-IBA2 plasmid (IBA Life SciencesGermany) under the control of T7 promoter.

Transformation. E. coli BL21 DE3 (from Life Technologies) cells weretransformed with the expression vector comprising the DNA encoding thewild-type or variant α-hemolysin using techniques well known in the art.Briefly, the cells were thawed on ice (if frozen). Next, the desired DNA(in a suitable vector/plasmid) was added directly into the competentcells (should not exceed 5% of that of the competent cells) and mixed byflicking the tube. The tubes were placed on ice for 20 minutes. Next,the cells were placed in a 42° C. water bath for 45 seconds withoutmixing, followed by placing the tubes on ice for 2 min. The cells werethen transferred to a 15 ml sterilized culture tube containing 0.9 ml ofSOC medium (pre-warmed at room temperature) and cultured at 37° C. for 1hr in a shaker. Finally, an aliquot of the cells was spread onto a LBagar plate containing the appropriate antibiotic and the platesincubated at 37° C. overnight.

Protein Expression. Following transformation, colonies were picked andinoculated into a small volume (e.g., 3 ml) of growth medium (e.g., LBbroth) containing the appropriate antibiotic with shaking at 37° C.,overnight.

The next morning, 1 ml of the overnight culture was transferred to a new100 ml of autoinduction medium, e.g., Magic Media (Life Technologies)containing an appropriate antibiotic to select the expression plasmid.The culture was grown with shaking at 25° C. approximately 16 hrs butthis depended on the expression plasmids. Cells were harvested bycentrifugation at 3,000 g for 20 min at 4° C. and stored at −80° C.until used.

Purification. Cells were lysed via sonication. The alpha-hemolysin waspurified to homogeneity by affinity column chromatography.

Example 2 T12 and/or N17 Variants

The following example details the introduction of a mutation at adesired residue.

Mutations. Site-directed mutagenesis was carried out using a QuikChangeMulti Site-Directed Mutagenesis kit (Stratagene, La Jolla, Calif.) toprepare the T12 and/or N17 variants of SEQ ID NO:3.

The variants were expressed and purified as in Example 1.

Example 3 Assembly of Nanopore

This example describes the assembly of a nanopore comprising six a-HLvariant subunits and one wild-type subunit.

The wild-type a-HL was expressed as described in Example 1 with SpyTagand a HisTag and purified on a cobalt affinity column using a cobaltelution buffer (200 mM NaCl, 300 mM imidazole, 50 mM Tris, pH 8). Thedesired a-HL variant was expressed as described in Example 1 with aStrepTag and purified using a Streptactin affinity column on the fastprotein liquid chromatography (FPLC) using an elution buffer (50 mMtris, 5 mM desthiobiotin, 200 mM NaCl, pH 8). The proteins were storedat 4° C. if used within 5 days, otherwise 8% trehalose was added and theproteins were stored at −80° C.

Using approximately 20 mg of total protein, the wild-type a-HL anddesired a-HL variant solutions were mixed together at the 1:6 ratio.Diphytanoylphosphatidylcholine (DPhPC) lipid was solubilized in 50 mMTris, 200 mM NaCl, pH 8 or 150 mM KCI, 30mM HEPES, pH 7.5 to a finalconcentration of 50 mg/ml and added to the mixture of a-HL monomers to afinal concentration of 5 mg/ml. The mixture of the a-HL monomers wasincubated at 40° C. for at least 10 min. The lipid hemolysin mixture wasapplied to a size-exclusion chromatography column to separate the lipidfrom the oligomerized proteins.

Example 4 Attachment of a Polymerase

This example provides for the attachment of a polymerase to a nanopore.

The polymerase may be coupled to the nanopore by any suitable means.See, for example, PCT/US2013/068967 (published as WO2014/074727; GeniaTechnologies), PCT/US2005/009702 (published as WO2006/028508), andPCT/US2011/065640 (published as WO2012/083249; Columbia Univ).

The polymerase, e.g., phi29 DNA Polymerase, was coupled to a proteinnanopore (e.g., alpha-hemolysin), through a linker molecule.Specifically, the SpyTag and SpyCatcher system, that spontaneously formscovalent isopeptide linkages under physiological conditions was used.See, for example, Li et al, J Mol Biol. 2014 Jan. 23; 426(2):309-17.

The Sticky phi29 SpyCatcher HisTag was expressed according to Example 1and purified using a cobalt affinity column. The SpyCatcher polymeraseand the SpyTag oligomerized protein were incubated overnight at 4° C. in3 mM SrCl₂. The 1:6-polymerase-template complex was then purified usingsize-exclusion chromatography.

Example 5 Activity of the Variants

This example shows the activity of the nanopores as provided by Examples3 and 4 (nanopores with an attached polymerase).

The wild-type and variant nanopores were assayed to determine the effectof a mutation at one or more positions. The assay was designed tomeasure the time it takes to capture a tagged molecule by a DNApolymerase attached to the nanopore using alternating voltages, i.e.,squarewaves.

The bilayers were formed and pores were inserted as described inPCT/US14/61853 filed 23 Oct. 2014. The nanopore device (or sensor) usedto detect a molecule (and/or sequence a nucleic acid) was set-up asdescribed in WO2013/123450.

To measure the time it takes to capture a tagged nucleotide by a DNApolymerase in our sequencing complex we have devised an assay that usesalternating positive and negative voltages (squarewaves) to determinethe amount of time this takes. Our sequencing complex is comprised of aprotein nanopore (αHL), which is attached to a single DNA polymerase(see Example 4). The tagged nucleotides are negatively charged, and aretherefore attracted to the nanopore when the voltage applied is positivein nature, and repelled when the voltage applied to the nanoporesequencing complex is negative. We can thus measure the time it takesfor a tag to thread into the pore by cycling the voltage betweenpositive and negative potentials and determine how much time thenanopore's current is unobstructed (open channel) verses when the tag isthreaded (reduced current flux).

To carry out this “time-to-thread” assay the Genia Sequencing device isused with a Genia Sequencing Chip. The electrodes are conditioned andphospholipid bilayers are established on the chip as explained inPCT/US2013/026514. Genia's sequencing complex is inserted to thebilayers following the protocol described in PCT/US2013/026514(published as WO2013/123450).

The time-to-thread data shown in this example was collected using abuffer system comprised of 20 mM HEPES pH 7.5, 300 mM KCl, 3 uM taggednucleotide, 3 mM Ca²⁺, with a voltage applied of +/−100 mV with a dutycycle of 5 Hz. After the data was collected it was analyzed forsquarewaves that showed the capture of a tagged nucleotide (threadedlevel) which lasted to the end of the positive portion of thesquarewave, and was followed by another tag capture on the subsequentsquarewave. The time-to-thread was measured by determining how long thesecond squarewave reported unobstructed open channel current. As anexample, if 10 consecutive squarewaves showed tagged nucleotide capturesthat lasted to the end of the positive portion of the squarewave thenthe time-to-thread parameter would be calculated from squarewaves 2-10(the first squarewave does not factor into the calculation because thepolymerase did not have a tag bound to it in the previous squarewave).These time-to-thread numbers were then collected for all of the pores inthe experiment and statistical parameters extracted from them (such as amean, median, standard deviation etc.).

Results are shown in FIGS. 1A-1B, 2A-2B, 3A-3B, 4A-4B, and 5A-5B.

Example 6 Breaking Mutations in Oligomerization-Deficient Subunits

This example shows that breaking mutations prevent or reduceself-oligomerization of the αHL monomer subunits.

Site-directed mutagenesis was carried out using a QuikChange MultiSite-Directed Mutagenesis kit (Stratagene, La Jolla, Calif.) tointroduce single amino acid mutations at each of first (site 1) andsecond (site 2) oligomerization domains of the α-hemolysin variantcomprising the N17K mutation described in Example 2.

DNA polynucleotides encoding hemolysin variant N17K monomers furthercomprising one of mutations: H35D, D24A+V26D+K37S, H35E, H35L, H35I,T233R+S99K, Y101D, Y101H, H35N, and T233R+S99K+D24A+V26D+K37S in thefirst or second oligomerization domain of the N17K variant, were clonedinto pPR-IBA2 plasmids, expressed in E. coli, and subsequently purifiedas described in Example 1.

The purified mutated N17K variant monomers were reconstituted in thepresence of lipid to determine the ability of each of the mutations ininhibiting the monomers from self-oligomerizing. 5 mg/mL DoPhPC Lipidwas added to protein at a concentration of 1 mg/ml, and the mixture wasincubated at 30° C. for 30 minutes. Liposomes were solubilized with 5%β-OG. The presence or absence of monomers and oligomers of each mutatedvariant was determined by subjecting the reconstituted mutated variantto SDS-PAGE gel electrophoresis. The results are shown in the gels ofFIGS. 8A and 8B, and lanes 4-7 of the gel shown in FIG. 13.

To test whether the breaking mutations could be rescued by wild-type αHLmonomer (Hemo M), Hemo M was added in a 1:1 ratio to monomers withmutations D24A+V26D+K37S, and T233R+S99K. The results shown in FIG. 9demonstrate that these breaking mutations could not be rescued bywild-type monomer, i.e., the unmutated amino acids on either the firstor second oligomerization domain on a wild-type monomer do not rescuethe interaction of monomers having the mutations shown.

Additionally, the relative mobility (Rf) of mutated variantmonomers/oligomers having mutations D24A+V26D+K37S, T233R+S99K, H35E,H35D, H35N, and H35L were determined.

The results of the Rf values are shown in Table 1 below. The unmutatedN17K variant monomer was determined to retain the ability toself-oligomerize, where 66.3% of the monomer was present as oligomers.

Taken together the data show that the breaking mutations made at one orboth of the oligomerization domains (sites 1 and 2) of the hemolysinvariant monomer, prevent or substantially reduce the ability of themonomer to self-oligomerize.

TABLE 1 Self-Oligomerization of mutated N17K variant monomers MUTANTSELF-OLIGOMER % SELF-OLIGOMER D24A + V26D + K37S − 0 T233R + S99K − 0H35E − 0 H35D − 0 H35N − 0 H35L + 2.8

Example 7 Purification Reconstitution of Oligomerized Dimer Concatemers

This example demonstrates the formation of oligomers resulting from theoligomerization of a subunit comprising a concatemer of two wild-typeαHL monomers.

A DNA polynucleotide encoding an αHL protein comprising a signalsequence, (peIB), a first αHL monomer, (Hemo 1), a linker (GS)5 (SEQ IDNO: 9), a second αHL monomer (Hemo 2), and a purification tag-attachmenttag (His6-SpyTag) (“His6” disclosed as SEQ ID NO: 10), was cloned intopPR-IBA2 plasmids, expressed in E.coli, and subsequently purified asdescribed in Example 1.

The purified concatemer dimer purifies as a single peak obtained by SEC(FIGS. 10A and 10B).

The purified concatemer was reconstituted in the presence of lipid byincubating the protein with DoPhPC lipid for 10 minutes at 37° C. Theliposomes were solubilized, and subjected to electrophoresis, asdescribed in Example 6.

FIG. 11 shows that the αHL concatemer of two monomers has the ability ofself-oligomerizing.

It is expected that introducing the breaking mutation, for example asthose described for the monomer in Example 6, will abolish the abilityof the dimer concatemer to self-oligomerize, and may subsequently beused to manage the stoichiometry and arrangement of αHL subunits intofunctional heptameric pores.

Example 8 Expression and Purification of Oligomerized Trimers andTetramer Concatemers

This example shows the expression and purification of concatemers ofthree and four αHL monomers.

Trimer and tetramer concatemer subunits of αHL monomers were expressedand purified as described for the dimer concatemer in Example 7. Thepolynucleotide encoding the trimer concatemer expresses the followingoligomerization subunit: (peIB), a first αHL monomer, (Hemo 1), a linker(GS)5 (SEQ ID NO: 9), a second αHL monomer (Hemo 2), a linker (GS)5 (SEQID NO: 9), a third αHL monomer (Hemo 3), and a StrepII tag.

The polynucleotide encoding the tetramer concatemer expresses thefollowing oligomerization subunit:

signal sequence (peIB), a first αHL monomer, (Hemo 1), a linker (GS)5(SEQ ID NO: 9), a second αHL monomer (Hemo 2), a linker (GS)5 (SEQ IDNO: 9), a third αHL monomer (Hemo 3), a linker (GS)5 (SEQ ID NO: 9), afourth αHL monomer (Hemo 4), and a His-SpyTag.

The concatemers were expressed in E. coli and purified as described inExample 1, and then were subjected to electrophoresis.

FIG. 12 shows that concatenated subunits of three and four linkedmonomers can be expressed and purified. The dimers and monomers seen onthe gel are the result of degradation that occurs during purification,and that can be minimized by introducing an affinity tag at theN-terminus of the concatemer in combination with a purification stepusing the N-terminal affinity tag.

Example 9 Functional Hetero-Oligomeric Alpha-Hemolysin HeptamericNanopores

To determine the ability of subunit concatemers to form functional αHLpores, a first αHL concatemer subunit of three monomers is combined inthe presence of lipid with a second αHL concatemer subunit of fourmonomers to provide a heptameric αHL pore.

A breaking mutation i.e., amino acid substitution, is introduced in eachof the first and second oligomerization domains of the trimer and oftetramer concatemer subunits. Additionally, a cognate and/or rescuemutation is introduced as an amino acid substitution in the firstoligomerization domain of the trimer and the tetramer concatemers.Mutations are generated using site-directed mutagenesis as described inExample 2. A polymerase is attached to each of the concatemer subunitsusing the method described in Example 4.

The activity of the nanopore is measured as described in Example 5.

The hetero-oligomeric α-hemolysin heptamer of two concatemer subunitsretains the ability to form a heptameric nanopore in a lipid bilayer.

Example 10 Cognate Mutations Enable Oligomerization of Subunits

To demonstrate the ability of mutations in first and secondoligomerization domains of αHL subunits, amino acid substitutions H351and Y101H were made respectively in the first and second oligomerizationdomains of the variant N17K αHL monomers.

The mutated variant monomers were expressed in bacteria and purified asdescribed in Example 6.

Next, the purified mutated N17K variant monomers were reconstituted inthe presence of lipid to determine the ability of the mutations in theoligomerization domains in inhibiting or enabling oligomerization of themutated variant monomers. 5 mg/mL DoPhPC Lipid was added to monomerprotein at a concentration of 1 mg/ml, and the mixture was incubated at30° C. for 30 minutes. Liposomes were solubilized with 5% β-OG. Thepresence or absence of monomers and oligomers of the mutated variant wasdetermined by subjecting the reconstituted mutated variant to SDS-PAGEgel electrophoresis. The results are shown in FIG. 13.

Lanes 5 shows that mutation H351 alone inhibits oligomerization of themutated variant monomers. Similarly, lane 7 shows that mutation Y101Halone also inhibits oligomerization, i.e., H351 and Y101H were shown tobe breaking mutations. However, when both mutations H351 and Y101H weremade on the variant monomers (lane 9), the ability of the mutatedvariant αHL monomers was restored, i.e., when paired, H351 and Y101H arecognate mutations that allow inter-subunit interaction and enableoligomerization of αHL monomers.

These data show that subunit interaction and oligomerization of αHLsubunits, e.g., monomers, can be controlled by pairing of mutations madein the first and second oligomerization domains of subunits.

Example 11 Temperature-Dependent Conversion of Breaking Mutations

Experiments were performed to identify breaking mutations andcorresponding rescue and/or cognate mutations that would enableoligomerization of subunits, Applicants unexpectedly discovered breakingmutations that can convert to their own cognate mutations at highertemperatures.

αHL monomers having an H35G mutation in the first oligomerizationdomain, were reconstituted in lipid at 25° C. and at 37° C.Reconstitution was performed as described in Example 10.

The results are shown in FIG. 14. Lane 2 shows that in the presence oflipid, monomers having the H35G mutation does not oligomerize at 25° C.Therefore, lane 2 shows that the H35G mutation is a breaking mutationthat inhibits inter-subunit interaction, and oligomerization of themutated subunits. However, as shown in lane 4, when the same mutatedmonomers were reconstituted in lipid at 37° C., oligomerization of thesubunits was observed.

These data demonstrate that at higher temperatures, e.g., 37° C. versus25° C., subunits having the H35G breaking mutation can oligomerize.

Therefore, in addition to pairing breaking mutations with cognate and/orrescue mutations to enable oligomerization of subunits, breakingmutations can be converted to their own cognate mutations and enableoligomerization.

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application andscope of the appended claims. All publications, patents, and patentapplications cited herein are hereby incorporated by reference in theirentireties for all purposes.

SEQUENCE LISTING FREE TEXT (WT αHL DNA) SEQ ID NO: 1ATGGCAGATC TCGATCCCGC GAAATTAATA CGACTCACTA TAGGGAGGCC 50ACAACGGTTT CCCTCTAGAA ATAATTTTGT TTAACTTTAA GAAGGAGATA 100TACAAATGGA TTCAGATATT AATATTAAAA CAGGTACAAC AGATATTGGT 150TCAAATACAA CAGTAAAAAC TGGTGATTTA GTAACTTATG ATAAAGAAAA 200TGGTATGCAT AAAAAAGTAT TTTATTCTTT TATTGATGAT AAAAATCATA 250ATAAAAAATT GTTAGTTATT CGTACAAAAG GTACTATTGC AGGTCAATAT 300AGAGTATATA GTGAAGAAGG TGCTAATAAA AGTGGTTTAG CATGGCCATC 350TGCTTTTAAA GTTCAATTAC AATTACCTGA TAATGAAGTA GCACAAATTT 400CAGATTATTA TCCACGTAAT AGTATTGATA CAAAAGAATA TATGTCAACA 450TTAACTTATG GTTTTAATGG TAATGTAACA GGTGATGATA CTGGTAAAAT 500TGGTGGTTTA ATTGGTGCTA ATGTTTCAAT TGGTCATACA TTAAAATATG 550TACAACCAGA TTTTAAAACA ATTTTAGAAA GTCCTACTGA TAAAAAAGTT 600GGTTGGAAAG TAATTTTTAA TAATATGGTT AATCAAAATT GGGGTCCTTA 650TGATCGTGAT AGTTGGAATC CTGTATATGG TAATCAATTA TTTATGAAAA 700CAAGAAATGG TTCTATGAAA GCAGCTGATA ATTTCTTAGA TCCAAATAAA 750GCATCAAGTT TATTATCTTC AGGTTTTTCT CCTGATTTTG CAACAGTTAT 800TACTATGGAT AGAAAAGCAT CAAAACAACA AACAAATATT GATGTTATTT 850ATGAACGTGT AAGAGATGAT TATCAATTAC ATTGGACATC AACTAATTGG 900AAAGGTACAA ATACTAAAGA TAAATGGACA GATAGAAGTT CAGAAAGATA 950TAAAATTGAT TGGGAAAAAG AAGAAATGAC AAATGGTCTC AGCGCTTGGA 1000GCCACCCGCA GTTCGAAAAA TAA 1023(WT αHL amino acids) [as expressed in E. coli] SEQ ID NO: 2MADSDINIKT GTTDIGSNTT VKTGDLVTYD KENGMHKKVF YSFIDDKNHN 50KKLLVIRTKG TIAGQYRVYS EEGANKSGLA WPSAFKVQLQ LPDNEVAQIS 100DYYPRNSIDT KEYMSTLTYG FNGNVTGDDT GKIGGLIGAN VSIGHTLKYV 150QPDFKTILES PTDKKVGWKV IFNNMVNQNW GPYDRDSWNP VYGNQLFMKT 200RNGSMKAADN FLDPNKASSL LSSGFSPDFA TVITMDRKAS KQQTNIDVIY 250ERVRDDYQLH WTSTNWKGTN TKDKWTDRSS ERYKIDWEKE EMTNGLSAWS 300 HPQFEK 306(Mature WT αHL sequence for numbering) SEQ ID NO: 3ADSDINIKTG TTDIGSNTTV KTGDLVTYDK ENGMHKKVFY SFIDDKNHNK 50KLLVIRTKGT IAGQYRVYSE EGANKSGLAW PSAFKVQLQL PDNEVAQISD 100YYPRNSIDTK EYMSTLTYGF NGNVTGDDTG KIGGLIGANV SIGHTLKYVQ 150PDFKTILESP TDKKVGWKVI FNNMVNQNWG PYDRDSWNPV YGNQLFMKTR 200NGSMKAADNF LDPNKASSLL SSGFSPDFAT VITMDRKASK QQTNIDVIYE 250RVRDDYQLHW TSTNWKGTNT KDKWTDRSSE RYKIDWEKEE MTNGLSAWSH 300 PQFEK 305(N17K αHL amino acids) SEQ ID NO: 4 ADSDINIKTG TTDIGS KTTV KTGDLVTYDK ENGMHKKVFY SFIDDKNHNK 50KLLVIRTKGT IAGQYRVYSE EGANKSGLAW PSAFKVQLQL PDNEVAQISD 100YYPRNSIDTK EYMSTLTYGF NGNVTGDDTG KIGGLIGANV SIGHTLKYVQ 150PDFKTILESP TDKKVGWKVI FNNMVNQNWG PYDRDSWNPV YGNQLFMKTR 200NGSMKAADNF LDPNKASSLL SSGFSPDFAT VITMDRKASK QQTNIDVIYE 250RVRDDYQLHW TSTNWKGTNT KDKWTDRSSE RYKIDWEKEE MTNGLSAWSH 300 PQFEK 305(N17R αHL amino acids) SEQ ID NO: 5 ADSDINIKTG TTDIGS RTTV KTGDLVTYDK ENGMHKKVFY SFIDDKNHNK 50KLLVIRTKGT IAGQYRVYSE EGANKSGLAW PSAFKVQLQL PDNEVAQISD 100YYPRNSIDTK EYMSTLTYGF NGNVTGDDTG KIGGLIGANV SIGHTLKYVQ 150PDFKTILESP TDKKVGWKVI FNNMVNQNWG PYDRDSWNPV YGNQLFMKTR 200NGSMKAADNF LDPNKASSLL SSGFSPDFAT VITMDRKASK QQTNIDVIYE 250RVRDDYQLHW TSTNWKGTNT KDKWTDRSSE RYKIDWEKEE MTNGLSAWSH 300 PQFEK 305(T12K αHL amino acids) SEQ ID NO: 6 ADSDINIKTG T KDIGSNTTV KTGDLVTYDK ENGMHKKVFY SFIDDKNHNK 50KLLVIRTKGT IAGQYRVYSE EGANKSGLAW PSAFKVQLQL PDNEVAQISD 100YYPRNSIDTK EYMSTLTYGF NGNVTGDDTG KIGGLIGANV SIGHTLKYVQ 150PDFKTILESP TDKKVGWKVI FNNMVNQNWG PYDRDSWNPV YGNQLFMKTR 200NGSMKAADNF LDPNKASSLL SSGFSPDFAT VITMDRKASK QQTNIDVIYE 250RVRDDYQLHW TSTNWKGTNT KDKWTDRSSE RYKIDWEKEE MTNGLSAWSH 300 PQFEK 305(T12R αHL amino acids) SEQ ID NO: 7 ADSDINIKTG T RDIGSNTTV KTGDLVTYDK ENGMHKKVFY SFIDDKNHNK 50KLLVIRTKGT IAGQYRVYSE EGANKSGLAW PSAFKVQLQL PDNEVAQISD 100YYPRNSIDTK EYMSTLTYGF NGNVTGDDTG KIGGLIGANV SIGHTLKYVQ 150PDFKTILESP TDKKVGWKVI FNNMVNQNWG PYDRDSWNPV YGNQLFMKTR 200NGSMKAADNF LDPNKASSLL SSGFSPDFAT VITMDRKASK QQTNIDVIYE 250RVRDDYQLHW TSTNWKGTNT KDKWTDRSSE RYKIDWEKEE MTNGLSAWSH 300 PQFEK 305(Mature WT aHL; AAA26598) SEQ ID NO: 8ADSDINIKTG TTDIGSNTTV KTGDLVTYDK ENGMHKKVFY SFIDDKNHNK 50KLLVIRTKGT IAGQYRVYSE EGANKSGLAW PSAFKVQLQL PDNEVAQISD 100YYPRNSIDTK EYMSTLTYGF NGNVTGDDTG KIGGLIGANV SIGHTLKYVQ 150PDFKTILESP TDKKVGWKVI FNNMVNQNWG PYDRDSWNPV YGNQLFMKTR 200NGSMKAADNF LDPNKASSLL SSGFSPDFAT VITMDRKASK QQTNIDVIYE 250RVRDDYQLHW TSTNWKGTNT KDKWTDRSSE RYKIDWEKEE MTN 293

CITATION LIST Patent Literature

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Non-Patent Literature

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The entirety of each patent, patent application, publication, document,GENBANK sequence, website and other published material referenced hereinhereby is incorporated by reference, including all tables, drawings, andfigures. All patents and publications are herein incorporated byreference to the same extent as if each was specifically andindividually indicated to be incorporated by reference. Citation of theabove patents, patent applications, publications and documents is not anadmission that any of the foregoing is pertinent prior art, nor does itconstitute any admission as to the contents or date of thesepublications or documents. All patents and publications mentioned hereinare indicative of the skill levels of those of ordinary skill in the artto which the invention pertains.

What is claimed is:
 1. An isolated polypeptide comprising one or morealpha-hemolysin monomers, wherein at least one of the alpha-hemolysinmonomers comprises an amino acid sequence having at least 85% sequenceidentity to SEQ ID NO: 3 and an H35I substitution, a Y101H substitution,or a combination thereof of SEQ ID NO:
 3. 2. The isolated polypeptide ofclaim 1, wherein one or more of the alpha-hemolysin monomers furthercomprise a time-to-thread (TTT) substitution at a position correspondingto one or both of position 12 and position 17 of SEQ ID NO:
 3. 3. Theisolated polypeptide of claim 2, wherein the TTT substitution isselected from the group consisting of T12K, T12R, N17K, and N17R.
 4. Theisolated polypeptide of claim 1, wherein the isolated polypeptidecomprises a single alpha-hemolysin monomer.
 5. The isolated polypeptideof claim 1, wherein the isolated polypeptide comprises at least twoalpha-hemolysin monomers and wherein each monomer of the polypeptide isseparated from each adjacent monomer in the polypeptide by a flexiblelinker.
 6. The isolated polypeptide of claim 1, wherein the isolatedpolypeptide comprises at least two but no more than sevenalpha-hemolysin monomers.
 7. The isolated polypeptide of claim 1,wherein at least one of the alpha-hemolysin monomers further comprisesan attachment component.
 8. The isolated polypeptide of claim 7, whereinthe attachment component comprises a SpyTag or SpyCatcher peptide. 9.The isolated polypeptide of claim 7, wherein the attachment componentjoins the alpha-hemolysin monomer to a DNA polymerase, an RNApolymerase, or a reverse transcriptase.
 10. A heptameric pore complexcomprising seven alpha-hemolysin monomers, wherein at least one of thealpha-hemolysin monomers comprises an amino acid sequence having atleast 85% sequence identity to SEQ ID NO: 3 and an H35I substitution, aY101H substitution, or a combination thereof of SEQ ID NO:
 3. 11. Theheptameric pore complex of claim 10, further comprising a polymeraselinked to one of the alpha-hemolysin monomers.
 12. The heptameric porecomplex of claim 11, wherein the polymerase is covalently linked to thealpha-hemolysin monomer.
 13. The heptameric pore complex of claim 12,wherein the covalent linkage comprises a SpyTag or SpyCatcher peptide.14. The heptameric pore complex of claim 10, wherein one or more of thealpha-hemolysin monomers further comprises a time-to-thread (TTT)substitution at a position corresponding to one or both of position 12and position 17 of SEQ ID NO:
 3. 15. The heptameric pore complex ofclaim 14, wherein the TTT substitution is selected from the groupconsisting of T12K, T12R, N17K, and N17R.
 16. The heptameric porecomplex of claim 10, wherein each alpha-hemolysin monomer of theheptameric pore complex is disposed within a separate polypeptide fromthe other alpha-hemolysin monomers of the heptameric pore complex. 17.The heptameric pore complex of claim 10, wherein from two to seven ofthe alpha-hemolysin monomers of the heptameric pore complex are disposedwithin a single polypeptide.
 18. A hetero-oligomeric α-hemolysin (αHL)heptamer, comprising at least one preceding and at least one followingsubunit, each subunit comprising at least one αHL monomer and/or atleast one polypeptide comprising concatenated αHL monomers, wherein theat least one αHL monomer and/or the at least one polypeptide comprisingconcatenated αHL monomers comprises a mutation corresponding to H35Iand/or Y101H of SEQ ID NO:3; and wherein the heptamer comprises exactly7 αHL monomers, each αHL monomer comprising a first oligomerizationdomain and a second oligomerization domain, the first oligomerizationdomain of each αHL monomer being linked to the second oligomerizationdomain of a preceding αHL monomer and the second oligomerization domainof each αHL monomer being linked to the first oligomerization domain ofa following αHL monomer.
 19. The hetero-oligomeric αHL heptamer of claim18, wherein at least one αHL monomer further comprises a time-to-thread(TTT) substitution at a position corresponding to one or both ofposition 12 and position 17 of SEQ ID NO: 3 and wherein
 20. Thehetero-oligomeric αHL heptamer of claim 19, wherein the TTT substitutionis selected from the group consisting of T12K, T12R, N17K, and N17R.